Blocking group 2 innate lymphoid cell activation and macrophage M2 polarization: potential therapeutic mechanisms in ovalbumin-induced allergic asthma by calycosin

Background Calycosin, a flavonoid compound extracted from Astragalus membranaceus, has shown anti-asthma benefits in house dust mite-induced asthma. Recent studies have suggested that innate-type cells, including group 2 innate lymphoid cells (ILC2s) and macrophages, serve as incentives for type 2 immunity and targets for drug development in asthma. This work focuses on the effects of calycosin on the dysregulated ILC2s and macrophages in allergic asthma. Methods In vivo, the asthmatic mouse model was established with ovalbumin (OVA) sensitization and challenge, and calycosin was intraperitoneally administered at doses of 20 and 40 mg/kg. In vivo, mouse primary ILC2s were stimulated with interleukin (IL)-33 and mouse RAW264.7 macrophages were stimulated with IL-4 and IL-13 to establish the cell models. Cells were treated with calycosin at doses of 5 and 10 µM. Results In vivo, we observed significantly reduced numbers of eosinophils, neutrophils, monocyte macrophages and lymphocytes in the bronchoalveolar lavage fluid (BALF) of OVA-exposed mice with 40 mg/kg calycosin. Histopathological assessment showed that calycosin inhibited the airway inflammation and remodeling caused by OVA. Calycosin markedly decreased the up-regulated IL-4, IL-5, IL-13, IL-33, and suppression tumorigenicity 2 (ST2) induced by OVA in BALF and/or lung tissues of asthmatic mice. Calycosin repressed the augment of arginase 1 (ARG1), IL-10, chitinase-like 3 (YM1) and mannose receptor C-type 1 (MRC1) levels in the lung tissues of asthmatic mice. In vivo, calycosin inhibited the IL-33-induced activation as well as the increase of IL-4, IL-5, IL-13 and ST2 in ILC2s. Calycosin also repressed the increase of ARG1, IL-10, YM1 and MRC1 induced by IL-4 and IL-13 in RAW264.7 macrophages. In addition, we found that these changes were more significant in 40 mg/kg calycosin treatment than 20 mg/kg calycosin. Conclusions Collectively, this study showed that calycosin might attenuate OVA-induced airway inflammation and remodeling in asthmatic mice via preventing ILC2 activation and macrophage M2 polarization. Our study might contribute to further study of asthmatic therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s40360-024-00751-9.


Introduction
Asthma is a common respiratory disease characterized by chronic airway inflammation, bronchial hyperresponsiveness, excessive mucus secretion, and airway remodeling [1][2][3].Clinically, dyspnea, wheeze, cough and chest tightness often occur in patients with asthma [1].In addition, asthma causes severe economic and family burdens due to the high cost of treatment [4].Therefore, it is absolutely imperative that effective drugs for asthma and the underlying mechanisms are further explored and clarified.Based on the previous immunological studies of asthma, the endotypes of asthma are divided into type 2 immune responses and non-type 2 immune responses [5,6].The type 2 immune response consists of T helper 2 (Th2) cells and group 2 innate lymphoid cells (ILC2s).In the study, we applied ILC2s to explore the mechanisms of calycosin for asthma treatment.
ILC2s belong to the family of innate lymphoid cells (ILCs), which are effector cells lacking T cell and B cell antigen receptors and involved in tissue remodeling and innate immunity [7].ILCs have been classified into ILC1s, ILC2s and ILC3s on the basis of the function of cytokine production upon activation [8].ILC2s have been extensively applied in the research associated with asthma [9,10].Multiple studies have stated that ILC2s are elevated in the blood of asthmatic patients [11][12][13].Epithelial cell-derived cytokines, such as interleukin (IL)-25 and IL-33, are capable of stimulating ILC2s to produce T-helper (Th) type 2 cytokines, including IL-4, IL-5 and IL-13, thereby facilitating the pathogenesis of asthma [14][15][16][17][18].The concentrations of IL-33 are increased in the bronchoalveolar lavage fluid (BALF) of asthmatic patients, suggesting that IL-33 is closely correlated with asthmatic severity [14,19,20].Suppression tumorigenicity 2 (ST2) is known as the receptor of IL-33 and has a high expression in macrophages, natural killer cells, and ILC2s [21,22].A CpG-oligodeoxynucleotide alleviates Th2/Th17 inflammatory response, airway inflammation and remodeling in mice with smoke-related asthma by downregulating the IL-33/ST2 axis [23].IL-33/ST2 signaling results in the development of airway hyperresponsiveness in the peripheral lung in house dust miteinduced asthmatic mice [24].Therefore, targeting the inhibition of ILC2 activation by the IL-33/ST2 pathway prevents asthma, which is of great significance for studying therapeutic strategies for asthma.
The development of alternatively activated M2 macrophages is capable of being driven by ILC2s [25].
Macrophages are innate immune cells that participate in initiating an immune response [26].Macrophages are differentiated into classically activated M1 or alternatively activated M2 macrophages [27].Increased M2-phenotype macrophages are found in BALF and airway wall tissues in bronchial asthmatic patients [28][29][30].MicroRNA-21 antagonism decreases airway hyperresponsiveness and airway remodeling process by suppressing alveolar M2 macrophage polarization in an ovalbumin (OVA)-induced allergic asthma mouse [31].Suppression of M2 macrophage polarization by 2-chloroadenosine is accompanied by the reduction in airway inflammation and remodeling in asthmatic mice [32].These findings indicate that drug intervention targeting the inhibition of M2 macrophage polarization may be an effective approach for asthmatic treatment.
Overall, it is vital for asthma treatment to explore novel and effective drugs that target the inhibition of ILC2 activation and M2 macrophage polarization.A previous study has shown that calycosin mitigates pulmonary inflammation and hyper-responsiveness by protecting epithelial integrity via regulating the G protein-coupled estrogen receptor in a house dust mite-induced mouse model of allergic asthma [33].Calycosin (7,3'dihydroxy-4'-methoxyisoflavone) is a flavonoid component of Astragalus membranaceus, which has a great effect on the treatment of the recurrence of allergic diseases [33,34].Calycosin has been proven to possess the properties of immune regulation and anti-inflammation [35].Calycosin ameliorates renal injury in diabetic rats by inhibiting of the mRNA expression of both IL-33 and ST2 [36].Hence, we conjectured that calycosin might be a potential targeted medicine for asthma by regulating ILC2 activation and M2 macrophage polarization.
Herein, an OVA-sensitized asthmatic mouse model was established to investigate the effects of calycosin.It was investigated that calycosin repressed ILC2 activation and macrophage M2 polarization, thereby ameliorating asthma.

Animal model
The animal model was established following previous literature [37].Female BALB/C mice (6-8 weeks) were obtained from Liaoning Changsheng Biotechnology Company Limited (China).For establishing a mouse model of asthma, the mice were divided into four groups, including sham, OVA, OVA + 20 mg/kg calycosin (OVA + 20Ca), and OVA + 40 mg/kg calycosin (OVA + 40Ca) groups.The mice in the OVA group were intraperitoneally injected with 100 µl phosphate-buffered saline (PBS) containing 100 µg OVA (A107820, Aladdin, Shanghai, China) and 4 mg aluminum hydroxide on days 0, 7, and 14.On days 21 to 26, the mice were treated with aerosolized 1% OVA for 30 min each day.20 and 40 mg/kg calycosin (≥ 98% purity, verified by High Performance Liquid Chromatography (HPLC), C 16 H 12 O 5 ; B20846, Yuanye, Shanghai, China) were administered to the mice in the OVA + 20Ca and OVA + 40Ca groups respectively by intraperitoneal injection at 1 h before atomization.The doses were selected because of the potential effectiveness in asthma according to the previous literatures [38][39][40].The mice in the sham group were given the same volume of PBS without OVA.Twentyfour hours after the last OVA challenge, the mice were euthanized by isoflurane inhalation (3% for induction and 1.5% for maintenance) and exsanguination from the inferior vena cava.Serum was collected.The lung tissues were stored at -70°C or fixed in 4% paraformaldehyde for further experiments.All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Heilongjiang University of Chinese Medicine (2022062703).All methods were conducted according to the relevant guidelines and regulations for handling laboratory animals.

Cell counts in the BALF
BALF was harvested from the mice.The total inflammatory cell counts including eosinophils, neutrophils, monocyte macrophages and lymphocytes were determined in the BALF by Giemsa (D010, Jiancheng Bioengineering Institute, Nanjing, China) staining.

Enzyme-linked immunosorbent assay (ELISA)
The levels of immunoglobulin E (IgE) in serum were examined on the basis of the Mouse IgE ELISA kit (Multisciences Biotech, Hangzhou, China).The levels of IL-4, IL-5, IL-13, IL-33 and IL-10 in the BALF or the cells were detected using the corresponding Mouse IL-4/ IL-5/IL-13/IL-33/IL-10 ELISA Kit (Multisciences Biotech, Hangzhou, China).The concentrations of arginase 1 (ARG1) in the BALF or RAW264.7 cells were detected by the Mouse ARG1 ELISA kit from Fine Biotech (Wuhan, China).
Additionally, PAS (DG0005, Leagene, Beijing, China) was used to stain the slices.The images were captured and observed at 200 × magnification by a microscope (Olympus, Tokyo, Japan).The degree of airway wall thickening and mucus hypersecretion was scored according to the previous study [47].

Statistical analysis
GraphPad Prism 8.0 software was utilized to analyze all the data, which were presented as mean ± SD.A one-way ANOVA with Tukey's test was performed for multiple group comparisons.Kruskal-Wallis non-parametric test was used to compare the pathology scores among multiple groups.Significance was set at p-value < 0.05.

Calycosin blocked macrophage M2 polarization in asthmatic mice
The protein levels of M2 macrophage markers ARG1 and IL-10 were assessed in the BALF by ELISA (Fig. 3A and  B).The mRNA levels of YM1 and MRC1 were detected in the lung tissues by RT-qPCR (Fig. 3C and D).OVA led to the up-regulation of these M2 macrophage markers.However, the levels of these markers were declined after the administration of calycosin (p < 0.05, Fig. 3A-D).Calycosin obstructed the co-localization of MRC1 and F4/80 induced by OVA in asthmatic mice (p < 0.05, Fig. 3E).Therefore, calycosin attenuated macrophage M2 polarization in the mice treated with asthma.

Calycosin inhibited IL-33-induced ILC2 activation in ILC2s
To further investigate the roles of calycosin in ILC2 activation, we isolated ILC2s from the lung tissues of BALB/C mice.The ILC2s were identified by detecting the cell surface molecules, including CD45, CD25, ST2 and c-Kit (Figure S1A-D).IL-33 treatment increased the viability in the isolated ILC2s.Calycosin reversed the effects (p < 0.01, Fig. 4A).On the one hand, calycosin decreased the increase in ST2 protein levels induced by IL-33 in ILC2s (Fig. 4B).On the other hand, calycosin suppressed the enhanced release of IL-4, IL-5 and IL-13 in IL-33-treated ILC2s (p < 0.05, Fig. 4C-E).It was suggested that ILC2 activation was repressed by calycosin in ILC2s.

Discussions
In this study, we established an asthmatic mouse model using OVA.Calycosin inhibited the number of inflammatory cells in asthmatic mice.Additionally, calycosin exerted inhibitory effects on the enhanced inflammatory responses that were caused by OVA in mice.Besides, we elucidated that calycosin markedly alleviated OVA-challenged asthma in mice through obstructing ILC2 activation and macrophage M2 polarization via measuring the associated markers.
ILC2s, corresponding to Th2 cells in adaptive immunity, have been confirmed to play a dominant role in allergic diseases such as asthma [50].The patients with asthma have more ILC2s in the blood and sputum [12,13,51].IL-33, which serves as a Th2 cytokine activator in ILC2s, is closely involved in inflammatory conditions [52].When subjects with asthma have been challenged with an inhalational allergen, IL-33 is ascending in the bronchial epithelium [53,54].IL-33 activates lung ILC2s by binding its receptor ST2, which is highly expressed in ILC2s in asthmatic mice [55,56].It has been discovered that the IL-33/ST2 pathway plays a significant role in asthma [57].In the previous study, up-regulated IL-33 and ST2 expression in OVA-sensitized lungs are reduced through salidroside administration, reflecting that salidroside treats allergic asthma by repressing lung ILC2 activation via targeting the IL-33/ST2 axis [45].Osthole attenuates OVA-caused lung inflammation via blocking IL-33/ST2 signaling in asthmatic mice [58].In line with these studies above, the levels of IL-33 and ST2 were both up-regulated in the lung tissues in OVA-exposed asthmatic mice.However, we found that calycosin downregulated the levels of IL-33, and ST2 to diminish ILC2 activation induced by OVA in mice.In addition, calycosin inhibited IL-33-induced the increase of ST2 to suppress ILC2 activation in vitro.These results indicated that calycosin targeted the IL-33/ST2 axis to suppress ILC2 activation, thereby serving as a protective medicine in asthma.
IL-33 administration stimulates ILC2s to produce IL-4 and IL-13, which are capable of polarizing M2 macrophages [59][60][61].More and more researchers have observed that M2 macrophage polarization is closely linked with the development of asthma [28,29,62].Moreira and colleagues have discovered that serum amyloid P prevents airway resistance to methacholine, inflammation, and remodeling in asthmatic mice with Aspergillus fumigatus allergy, which may involve the inhibition of the M2 activation of macrophages [63].Decreased M2 macrophage polarization suppresses P < 0.01, ### P < 0.001, compared with the OVA group."Ca" represents calycosin asthmatic airway inflammation in the microRNA-21-deficiency lung tissues in OVA-induced mice [31].Histone deacetylase 8 inhibitor ameliorates airway hyper-responsiveness and allergic airway inflammation in mice with allergic asthma through declining M2 macrophage polarization [64].These studies have suggested that inhibiting M2 macrophage polarization is significant for asthma treatment.In our study, the levels of ARG1, IL-10, YM1, MRC1 and F4/80 were used to reflect the levels of M2-polarized macrophages according to the previous literatures [65][66][67][68][69].Our study manifested that calycosin blocked OVA-induced M2 macrophage polarization in mice and repressed M2 macrophage polarization driven by IL-4 and IL-13 in RAW264.7 macrophages.It was thus clear that the protective role of calycosin in asthma might be due to the suppression of M2 macrophage polarization.
Calycosin is a typical phytoestrogen extracted from the root of Astragalus membranaceus that is effective in treating cardiovascular disease, nephritis, diabetes mellitus, childhood asthma, allergic rhinitis and cancers [39].Calycosin has been reported to possess multiple biological activities, such as anti-oxidative stress, antiinflammatory, antibacterial, and neuroprotective effects [39].Calycosin reduces diabetes-induced kidney inflammation through inhibiting phosphorylation of p65 in the nuclear factor-κB (NF-κB) signaling pathway [70,71].Calycosin suppresses autophagy and oxidative stress in chronic kidney disease skeletal muscle atrophy by regulating the AMPK/SKP2/CARM1 signaling pathway [72].Interestingly, previous studies have shown that calycosin suppresses IL-33 levels in various inflammatory disorders [36,73].Calycosin significantly attenuates renal injury in diabetic rats, involving inhibition of IL-33/ST2 axis signaling [36].In our study, calycosin was capable of reducing the levels of IL-33 and ST2 to inhibit ILC2 activation in asthma.Currently, it has not been reported that ILC2 activation and M2 macrophage polarization are involved in asthma therapy by calycosin.Our research might provide new insights for the treatment of asthma.
This study has the following limitations: Firstly, although we preliminarily investigated that calycosin might block ILC2 activation and M2 macrophage polarization to alleviate asthma in mice, there are no further in vivo experiments to prove the toxicity of calycosin on other cells.Thus, it is still necessary to further determine whether calycosin safely exerts its therapeutic effects in vivo.Secondly, the current researches have suggested that calycosin is almost nontoxic.However, excessively low or high doses may cause side effects in patients.It is of great significance to explore the optimal concentration of calycosin clinically in the future.Thirdly, although we found that the cytokines or markers, such as IL-33, ST2, IL-4 and MRC1, participated in the treatment of calycosin in asthma, the direct molecular mechanism of action is still unclear.We may use molecular docking and single cell RNA sequencing (scRNA-seq) to further investigate the potential molecular mechanism in asthma in the future.Fourthly, it is one-sided that RAW264.7 cells are used instead of primary cells.Although RAW264.7 cells have been directly applied to study the roles of macrophages in multiple studies, it is still necessary to verify the results in primary cells in the future for more rigorous and credible results.Finally, in vivo experiments should be further improved.We used IF to determine the expression of ST2 and CD45 to show the levels of ILC2s (ST2 + CD45+) and F4/80 and MRC1 to show the levels of M2 macrophages (F4/80 + MRC1+) in mouse lung tissues.However, it is significant to further investigate the number of ILC2 and M2 macrophages in fresh BALF and the phenotype changes in these two cell types after treatment with calycosin.

Conclusions
In summary, we found that calycosin protected asthmatic mice from inflammation injury by targeting the suppression of ILC2 activation and macrophage M2 polarization.Our study might provide a promising strategy for asthma therapy.